Project Summary Aging is the single largest risk factor for most chronic degenerative diseases, including cardiovascular, musculoskeletal and neurodegenerative dysfunctions, and age-associated diseases now represent the most rapidly growing unmet medical need in our society. Yet, while certain ?hallmarks of aging? have been defined, the critical molecular mediators that drive (and oppose) mammalian aging phenotypes have yet to be systematically explored, due in part to the technological challenges of applying classic functional genetic approaches, which require the laborious generation and aging(!) of complex gene-specific germline and conditional knockout alleles, to studies of aging physiology. As this substantial knowledge gap presents a significant impediment to developing new therapies for human aging pathologies, we aim in this project to establish a new, more facile approach to interrogating the genes and pathways that regulate mammalian organ function and repair throughout life. Our approach will take advantage of a unique in vivo genome editing system through which we can experimentally induce programmable mutations into the genomes of stem cells in the blood and skeletal muscle in intact animals, without the requirement for stem cell isolation and transplantation. By targeting endogenous stem cells, we allow for the maintenance and propagation of these mutations but circumvent the need to remove these cells their native biological niche, which can induce cellular stress and alter stem cell behavior by exposure to non-physiological ex vivo conditions. We also enable rapid testing of potential combinatorial gene effects, in multiple genetic backgrounds and in mice of various ages. Thus, our approach provides a more powerful, and higher throughput, view into the molecular effectors of organismal aging, which will allow us to test previously unapproachable hypotheses regarding the impact of somatic mutagenic events in aging organ systems and to identify novel regulators that may drive the precocious onset of, or mediate protection from, degenerative phenotypes across organ systems. Taken together, our work will yield new platform technologies for interrogating mammalian gene functions in vivo, new insights into fundamental mechanisms of aging physiology and regenerative biology, and new and potentially broadly useful methods for effecting genetic therapies in situ.